Effects of Ca2+ and Zn2+ on trifluoperazine-S100 proteins interactions: induced circular dichroism and fluorescence spectra.

Interactions of trifluoperazine (TFP) with S100 proteins, EF-hand type Ca2+-binding proteins, in the presence of Ca2+ and Zn2+ were studied with induced circular dichroism (CD) and fluorescence spectra. The positive CD bands of TFP were induced at around 265 nm by adding either S100a or S100a0 protein in the presence of Ca2+. No CD band of TFP was, however, induced by adding S100b protein in the presence of Ca2+. Addition of Zn2+ to the TFP/S100 protein solutions did not induce any CD band at all. The fluorescence intensity of 2-p-toluidinylnaphthalene 6-sulfonate (TNS) bound to S100a or S100a0 protein decreased by adding TFP in the presence of Ca2+, while that bound to S100b protein decreased by adding TFP in the presence of Zn2+, indicating that TFP binds to S100a protein and S100a0 protein in a Ca2+-dependent manner and to S100b protein in a Zn2+-dependent manner. From these results together with other experimental findings it was suggested that (1) TFP binds to S100a protein and S100a0 protein in the presence of Ca2+, with half-saturation points of 18 and 3 microM, respectively, (2) TFP binds to S100b protein only in the presence of Zn2+, (3) alpha-subunit of S100 protein binds to TFP specifically in a Ca2+-dependent manner and beta-subunit in a Zn2+-dependent manner.

Expression of cytochrome P-450d by Saccharomyces cerevisiae.

Rat liver microsomal cytochrome P-450d was abundantly expressed in the yeast Saccharomyces cerevisiae by using a yeast-Escherichia coli shuttle vector consisting of rat liver P-450d cDNA and yeast acid phosphatase promoter. The expressed cytochrome P-450d was immunologically crossed with rat liver P-450d. The hydroxylase activity of estra-1,3,5(10)-triene-3, 17 beta-diol was 11 nmol/min per nmol P-450d, which is comparable to that reported previously for rat liver P-450d. The expressed P-450d content was nearlyt 1% of total yeast protein as estimated from immunoblotting, hydroxylase activity and optical absorpton of the reduced CO form.

Interaction between chicken gizzard caldesmon and tropomyosin.

Chicken gizzard muscle caldesmon has been examined for ability to interact with tropomyosin from chicken gizzard muscle by using fluorescence enhancement of tropomyosin labeled with dansyl chloride (DNS) and affinity chromatography. The binding of caldesmon to tropomyosin was regulated by Ca2+ and calmodulin, i.e., at low ionic strength most of the caldesmon bound to tropomyosin-Sepharose 4B was co-eluted by adding calmodulin only in the presence of Ca2+, but not in its absence. This regulation by Ca2+ and calmodulin was also suggested by fluorescence measurements. Actin- and calmodulin-binding sites on the caldesmon molecule were located in the 38K fragment (Fujii, T., Imai, M., Rosenfeld, G.C., & Bryan, J. (1987) J. Biol. Chem. 262, 2757-2763). When 38K-enriched fraction was applied to the tropomyosin-Sepharose, the 38K fragment was retained by the column and could be eluted by adding Ca2+ and calmodulin.

Interaction of calponin with phospholipids.

The interaction between chicken gizzard calponin and phospholipids was examined by sedimentation assay and affinity chromatography. Calponin was sedimented with phosphatidylserine (PS) and phosphatidylinositol (PI) vesicles but not with phosphatidylcholine (PC) vesicles. The apparent Kd values of calponin to PS and PI were calculated to be 1.3 x 10(6) and 1.5 x 10(6) M-1, respectively. Domain mapping with chymotryptic digestion showed that the phospholipid-binding site resided within the N-terminal 22-kDa fragment, in which the bindings of actin, calmodulin, S100, and tropomyosin also occur. The amount of calponin bound to PS and PI vesicles decreased with increasing ionic strength or Ca2+ concentrations. The presence of MgCl2 was needed for the calponin-PS vesicle interaction. Calponin-binding proteins including actin, calmodulin, and S100 inhibited calponin binding to the phospholipid vesicles in a concentration-dependent manner, while tropomyosin had little effect on the binding. The inhibitory effects of calmodulin and S100 were found only in the presence of CaCl2. Neither caldesmon nor SM22 affected the binding.

Microtubule-associated proteins, MAP 1A and MAP 1B, interact with F-actin in vitro.

Microtubule-associated protein (MAP) 1 consisting of MAP 1A and 1B was purified from rat brain by the poly-L-aspartic acid (PLAA) method. We found that MAP 1 bound to F-actin in vitro up to a molar ratio of MAP 1 to actin monomers of 1:10. The apparent binding constant was about 2.7 x 10(7) M-1. In contrast to the binding of MAP 2 or tau to F-actin, the binding of MAP 1 to F-actin did not affect the low-shear viscosity of actin filaments. Binding experiments performed using fragments of MAP 1, obtained by chymotrypsin digestion, indicated that MAP 1 included binding domains to F-actin that were different from those in microtubules and also two light chains (31 and 29 kDa) that were cosedimented with F-actin as well as with microtubules.

In situ observation of bovine serum albumin-adsorbed stearic acid monolayer by Brewster angle microscopy.

The morphology of protein-adsorbed stearic acid monolayers containing a fluorescent probe (rhodamine B octadecylester perchloride: RBO) was observed by using fluorescence spectroscopy, fluorescence microscopy, and Brewster angle microscopy (BAM). The quenching of fluorescence of RBO was observed at the limiting area of the stearic acid monolayer. Thus, the fluorescence of RBO could be a good marker for packing of the matrix monolayer. On the other hand, the BAM image showed the morphology of not the matrix monolayer, but the adsorbed protein monolayer. Thus, the packing processes of the two could be distinguished. The present method is available for a protein that does not have visible absorption, such as bovine serum albumin (BSA). It is suggested that electrostatic interaction between matrix and protein molecules greatly affects the change in the morphology of the protein-adsorbed monolayer.

Morphology of a cytochrome c-adsorbed stearic acid monolayer on Brewster angle microscopy.

The morphologies of stearic acid and cytochrome c (cyt.c)-adsorbed stearic acid monolayers were investigated by Brewster angle microscopy (BAM) with various molecular areas of stearic acid. With an area of more than 0.38 nm2/molecule, many blight island domains and some bright circles were observed in the BAM image of the stearic acid monolayer. The blight site part became to occupy all the surface with compression, and then became more closely packed with an area of 0.22 nm2/molecule. On the other hand, a different BAM image was obtained for the cyt.c-adsorbed stearic acid monolayer, as follows: (i) a striped pattern was only observed in the presence of cyt.c; (ii) the number of bright circles in the presence of cyt.c was less than that in its absence. Furthermore, when a uniform BAM image was observed for the stearic acid monolayer with cyt.c, the intensity of the absorbance at 409 nm of cyt.c was the highest. By calculating the amount of cyt.c adsorbed on a stearic acid monolayer from the absorbance value, it was shown that cyt.c was most closely packed when an uniform BAM image was observed. These results suggest that the use of BAM and visible absorption spectroscopy together is useful for studying the morphology of a monolayer.

19F-n.m.r. study of trifluoperazine-S100 protein interaction: effects of Ca2+ and Zn2+.

19F-n.m.r. spectra were measured to investigate the effects of Ca2+ and Zn2+ on the interaction of trifluoperazine (TFP) with three S100 proteins. It was found that TFP binds to S100a and S100ao proteins irrespective of the presence of Ca2+ and Zn2+, while in the presence of Ca2+ the apparent affinity of TFP to the proteins was greater than that in its absence or in the presence of Zn2+. In contrast, the binding affinity of TRP to S100b protein in the presence and absence of metal ions was lower than to S100a and S100ao proteins. These results suggested that TFP binds to each S100 protein in two ways: one is Ca2(+)- or Zn2(+)-dependent specific manner and another is Ca2(+)- or Zn2(+)-independent non-specific manner.

Binding study of metal ions to S100 protein: 43Ca, 25Mg, 67Zn and 39K n.m.r.

The interactions of the S100 protein (S100) with metal cations such as Ca2+, Mg2+, Zn2+ and K+ were studied by the metal n.m.r. spectroscopy. The line widths of 43Ca, 25Mg, 67Zn and 39K n.m.r. markedly increased by adding all S100s. A broad 43Ca n.m.r. band of Ca(2+)-S100a solution was not affected by Zn2+ and K+, while it was greatly decreased by adding Mg2+. The 43Ca n.m.r. spectra of Ca(2+)-S100a0 and -S100b solutions consisted of two slow-exchangeable signals which corresponded to Ca2+ bound to two environmentally different sites of the S100a0. These two 43Ca n.m.r. signals were not affected by Zn2+ and K+. The line width of broad 25Mg n.m.r. band of the Mg(2+)-S100 solution greatly decreased by adding Ca2+, while it did not change by adding Zn2+ and K+. Further, the addition of Ca2+, Mg2+ and K+ did not affect the line width of the 67Zn n.m.r. of the Zn(2+)-S100 solutions. These findings suggest that: (1) Mg2+ binds to all S100s, and at least one of the Mg2+ binding sites of S100 molecule is the same as the Ca2+ binding site; (2) Zn2+ binds to S100s, although the binding site(s) is/are different from Ca(2+)- or Mg(2+)-binding site(s), and the environment of Zn2+ nuclei will not change even though Ca2+ binds to S100s.

Interaction of a protein-bound polysaccharide (PSK) with chicken gizzard caldesmon.

The interaction of a protein-bound polysaccharide (PSK) with caldesmon was studied in detail using sedimentation, low-shear viscometry, electron microscopy and affinity chromatography. The binding of caldesmon to F-actin was concentration-dependently inhibited by PSK. Bundle formation of actin filaments owing to caldesmon was also inhibited by PSK. Caldesmon bound to a PSK-Sepharose 4B column at low ionic strength was released at about 400 mM NaCl, whereas G-actin was not retained by the column. Treatment of caldesmon with chymotrypsin produced major fragments near 100, 80, 60, 38 and 25 kDa. In contrast, 60 and 25 kDa fragments were rarely formed by this treatment in the presence of PSK. Fragments of 80 and 38 kDa, major products produced by chymotrypsin, bound individually to a PSK-Sepharose column, indicating that caldesmon has at least two binding sites for PSK. Addition of calcium and calmodulin partially released caldesmon from actin filaments. PSK-dependent release of caldesmon was also observed in the presence of calcium and calmodulin.

Effects of calcium-binding proteins on histamine release from permeabilized rat peritoneal mast cells.

Among the calcium-binding proteins from chicken gizzard smooth muscle, smooth muscle calcium-binding protein 11 (SMCaBP 11), which is an EF-hand type calcium-binding protein, can prevent Ca2+ dependent histamine release from permeabilized rat peritoneal mast cells. This activity appears to be specific since other calcium-binding proteins (CaBPs), including h-calcimedin, l-calcimedin, calmodulin and S100, had no inhibitory action. Approximately 40% of the histamine release was inhibited by SMCaBP 11 at 48 nM and the half-maximal inhibition occurred at 20 nM. Immunological techniques revealed that permeabilized mast cells elicited by Ca2+ also released actin into the medium, but not tubulin. The addition of SMCaBP 11 prevented the secretion of actin from the mast cells, though the distribution of F-actin was only slightly affected. These findings suggest that SMCaBP 11 may modulate Ca2+ and actin-mediated exocytosis from permeabilized mast cells.

Structural and functional characterization of calelectrins from bovine liver.

Two Ca2(+)-dependent membrane-binding proteins with apparent molecular weights of 70000 (calelectrin70) and 32000 (calelectrin32) were isolated from bovine liver using phenyl-Sepharose affinity chromatography followed by diethylaminoethyl (DEAE)-cellulose and Ultrogel AcA44 chromatographies. Limited proteolysis and immunological analyses indicated that calelectrin32 was not a digested product from calelectrin70. Both calelectrins bound to phosphatidylserine and to calmodulin in a Ca2(+)-dependent manner. Circular dichroism studies showed that the apparent alpha-helical contents of calelectrin70 and calelectrin32 were 25 and 40%, respectively and they underwent Ca2(+)-dependent conformational changes. When the calelectrins were incubated with a brain microtubule preparation, they were phosphorylated by endogenous kinase(s) and phosphorylation occurred on serine residues. Moreover, calelectrin70 showed an inhibitory action on endogenous kinase activity in the presence of Ca2+.

Selective purification of microtubule-associated proteins 1 and 2 from rat brain using poly(L-aspartic acid).

A rapid and selective purification procedure for microtubule-associated protein (MAP) 1 and MAP 2 has been established. This procedure is based upon the fact that poly(L-aspartic acid) (PLAA) can specifically remove MAP 1 from microtubules polymerized by taxol (Nakamura et al., 1989, J. Biochem. 106, 93-97). MAP 1 released by PLAA was further purified by column chromatography on phosphocellulose and Bio-Gel A-15m. The purified MAP 1 contained MAPs 1A and 1 B. From microtubules devoid of MAP 1, MAP 2, consisting of MAPs 2A and 2B, could also be isolated by exposure to high ionic strength solutions in the presence of taxol without heat treatment. Both MAPs 1 and 2 cosedimented with microtubules consisting of purified tubulin.